Introduction to Flow Cytometry

1. Introduction to FlowCytometry IGC Workshop General Programme Module 1 – Cell Cycle Analysis using FACScan (Cláudia Bispo) Module 2 – Multicolor Analysis using CyAn ADP (Rui Gardner)

2. WhatisFlowCytometry? Flow Cytometry uic Introduction to FlowCytometry IGC Workshop Fundamentals ofFlowCytometry Rui Gardner IGC – April 2, 2013

3. Overview • Definitions: What is flow cytometry and what does it measure? • Fluidics: Hydrodynamic focusing, Flow Rate... • Optics: Light, fluorescence, emission and detection... • Electronics: pulse, data acquisition... • Analysis: Tips on Data handling 3

4. Resources • Purdue Univ. Cytometry Labs: all you need to know about Cytometry! • http://www.cyto.purdue.edu • Books: • Shapiro, H. “Practical Flow Cytometry”, 4ed, Online version. • http://PracticalFlowCytometry.com • Tutorials: • http://www.invitrogen.com/site/us/en/home/support/Tutorials.html • History of Flow Cytometry: • Shapiro, H. (2007) “Cytometry and Cytometers: Development and Growth”, • in Flow Cytometry with Plant Cells (Eds, J. Dolezel, J. Greilhuber, J. Suda), • WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4

5. Resources (Web Tools) BD Biosciences: http://www.bdbiosciences.com/research/multicolor/tools/index.jsp 5

6. Resources (Web Tools) Life Technologies: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Cell-and-Tissue-Analysis-Technical-Resources.html 6

7. Resources (Web Tools) eBioscience: http://www.ebioscience.com/resources.htm 7

8. Resources (Web Tools) Bioledgend: http://www.biolegend.com/webtoolstab 8

9. What is Flow Cytometry? Cytometry: Measurement of physical and chemical properties of cells. FlowCytometry: Characterization of cells flowing in a stream of fluid What is Flow Cytometry used for? Flow cytometry can be used to count large numbers of cells or particles based on size, internal compexity, phenotype, cellular state, cell function, DNA content, gene expression, and to quantify these same cellular properties at a single-cell level. Flow Cytometry is not equivalent to FACS: Fluorescence Activated Cell Sorting 9

10. KeyAdvantagesofFlowCytometry • Population data • Measure thousands of cells/particles per second • Measure multiple parameterssimultaneously. • Detection of extremely rare populations • Cell sorting • High purity, speed, andyield. Flow Cytometry does not... • Locate where a certain component is within a cell • Measure distribution of cellular components • Measure cell morphology 0.02%

11. Flow Cytometer in a nutshell Electronics Fluidics Optics converted to digital values that are processed and analyzed in a computer. Cells in suspension flow in single file through… an illuminated volume where they scatter light and emit fluorescence that is filtered, collected and… 11

12. Flow Cytometers Flow Cytometers Analyzers High Speed Cell Sorters MoFlo Legacy (BC) FACSAria (BD) MoFlo XDP (BC) MoFlo Astrios (BC) Influx (BD) FACSJazz (BD) FACS Vantage (BD) EPICS ALTRA (BC) Avalon (Propel Labs) Synergy sy3200 (sony-iCyt) SH800 (Sony) etc FACSscan (BD) FACSCalibur (BD) CyAn ADP (BC) LSR Fortessa (BD) FACSCanto (BD) EC800 (Sony-iCyt) Sony Spectral Analyzer (Sony) CyFlow Cube (Partec) MacsQuant (Miltenyi Biotec) Guava (Merck-Millipore) Gallios (BC) Accuri C6 (BD) Attune (Life Technologies) etc 12

13. Fluidics

14. Interrogation point 14

15. Simplified Schematics Flow Chamber Flow Chamber Sample Injected Sample Injected Sheath Flow Sheath Flow Sheath Flow Sheath Flow Hydrodynamic Focusing Laser Laser Flow Chamber in a BD FACSAria Cell Sorter Flow Chamber in a BD LSR Fortessa

16. Flow Rate High Flow Rate CyAn ADP Sheath Flow Sheath Flow Sample Flow Low Flow Rate Sheath Flow Sheath Flow Sample Flow LSR Fortessa Laser Laser Less cells pass per unit time More cells pass per unit time FACScan FACSCalibur 16

17. Flow Rate High Flow Rate Low Flow Rate Sheath Sheath Laser Laser Lower Power Lower Power High Power High Power Sample Sample • Samples should be run at the lowest flow rate (differential pressure) as possible. • If increasing flow rate has: • No impact on population distribution: Use high flow rate. • Changes population distribution: Use low flow rate and increase cellular concentration. 17

18. Exposure time Typical jet-in air cytometer Cell velocity is proportional to Sheath Pressure v ~ 5-30m/s Laser beam height ~ 5-20 µm Therefore exposure time ~ 10-7-10-5 s Typically, in sorters, sheath pressure can be reduced to increase exposure time, and therefore sensitivity and resolution of acquisition. Sheath pressure in Analyzers is usually fixed. 18

19. Optics

20. Excitation - Lasers 488 nm 442 Most common laser wavelengths available 20

21. Multiple Laser Systems Spatially Separated Laser Beams Laser Intercepts in Flow Cell FACSAria III (BD Biosciences) Laboratory of Molecular Immunology Institute Molecular Genetics, Academy of Sciences, Czech Republic http://mi.img.cas.cz/images/intercepts.jpg FACSAria III (BD Biosciences) Time delay between lasers must be determined for each instrument for a given sheath pressure. Changes in Sheath Pressure will impact measurements. 21

22. Emission - Light Scatter Light is reflected towards all directions Low angle: Forward Scatter (FSC) High angle: Side Scatter (SSC) 22

23. Forward Scatter (FSC) The magnitude of Forward Scatter is roughly proportional to the size of the cell. 23

24. Side Scatter (SSC) Side Scatter is caused by granulosity and structural complexity inside the cell. 24

25. (2D) Scatter Plot 25

26. Optics(Fluorescence)

27. Fluorescence (1) Excited State High Energy Absorbed Light Stokes Shift Emitted Light Absortion Emission Ground State Low Energy Fluorophore Energy Levels 27

28. Fluorescence(2) Stokes Shift High Energy 2 2 Absortion 1 1 3 Lower Wavelength Higher Wavelength 3 Low Energy Higher Frequency Lower Frequency Emission Higher Energy Lower Energy Fluorophore

29. Excitation-Emission Spectrum (2) Excitation maxima Emission maxima Each Fluorochrome has a specific Excitation and Emission Spectra

30. Excitation Spectrum (1) 480 nm 520 nm 550 nm 590 nm Excited 30

31. Excitation-Emission Spectrum (1) Excitation Emission Excitation max 550 nm

32. Excitation - Emission Excitation at different wavelengths decreases intensity of emission.

33. FluorescenceCharts

34. Fluorescence Spectra Viewers (Web Tools) • Spectra Viewers: • BD: http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/ • Life Technologies: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Labeling-Chemistry/Fluorescence-SpectraViewer.html • eBioscience: http://www.ebioscience.com/resources/fluorplan-spectra-viewer.htm • Bioledgend: http://www.biolegend.com/spectraanalyzer • UArizona: http://www.mcb.arizona.edu/ipc/fret/ • Evrogen: http://www.evrogen.com/spectra-viewer/viewer.shtml 34

35. Optics(FluorescentMarkers)

36. Reactive andConjugatedProbes CascadeBlue PacificBlue Pacific Orange AlexaDyes Luciferyellow NBD R-Phycoerythrin (PE) PE-Cy5 conjugates PE-Cy7 conjugates PE-TexasRed PerCP TruRed PerCP-Cy5.5 conjugate Fluorescein (FITC) BODIPY-FL TRITC X-Rhodamine XRITC LissamineRhodamine B Texas Red Allophycocyanin (APC) APC-Cy7 conjugates Etc… Immunohistochemistry Directmethod Indirectmethod Firstdye-coupledantibodyusingfluoresceinisothiocyanate (FITC) patentedbyJosephBurckhalterandRobertSeiwald, in 1960 Picturesadaptedfromwikipedia

37. Tandem Dyes PE-Alexa Fluor 700 PE-Cy5 PE-TxRed PerCP-Cy5.5 PE-Cy7 APC-Cy7 37

38. DNA andCellFunctionProbes

39. FluorescentProteins(1) GFP was first noted by Shimomura et al., in the jelly fish Aequoria Victoria, in 1962 and cloned 30 years later

40. FluorescentProteins(2) San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins Tsien Lab Roger Tsien, Nobel Lectures, 2009 http://www.tsienlab.ucsd.edu/HTML/Images/IMAGE - PLATE - Beach.jpg

41. WhatisFlowCytometry? Flow Cytometry uic Introduction to FlowCytometry IGC Workshop CoffeeBreak…!